We have a Zeiss LSM410 equipped with a 100x/1.3 oil immersion lens. When we
take x/y sections (20 sections, 0.5 micrometer apart) through a spherical
fluorescent bead (3.9 micrometer diameter, Multi Speck, Molecular Porbes,
M-7900) and apply built-in Zeiss 3D reconstruction functions, we obtain
something that looks like a football! The long axis of the football is
oriented along the z-axis and is about 1.5 x longer than the actual
diameter of the bead (z and x extension were both measured on an orthogonal
section). According to Molecular Porbes, the beads are embedded in a medium
with a refractive index matching the one of oil and sealed under a 1.5
coverslip. We therefore took the x/y sections using "1" as a refractive
index correction factor.

Our Zeiss representative tells us that what we see is "normal" and can't be
improved by adjusting the microscope. She suggest us to enter the z/x ratio
we determined (1.5 x, see above) as  refractive index correction factor
next time we take a stack of sections through a sample.

To us, the fluorescent beads appear to be a good model for a cell with a
fluorescent cytoplasm. Do we have to live with the fact, that images taken
under seemlingly optimal conditions are considerably distorted?

Thanks for comments and suggestions!

Benedikt Kost

Box 162
Lab of Plant Molecular Biology
The Rockefeller University
1230 York Avenue
New York, NY 10021-6399
USA